The invention relates to the field of histology.
In present day practice, the preparation of organic tissue samples and other material for transmission microscopy, both visible light and electron microscopy, is normally carried out by subjecting the sample to a series of chemical treatments culminating in the production of a solid block in which the sample is embedded. After the block is produced, thin sections of the sample (with the surrounding embedding material) are cut from the block and transferred to glass slides or other support. The embedding material may then be chemically removed and the tissue section stained with a variety of colored or fluorescent dyes, immunohistochemical stains, or subjected to in situ hybridization prior to examination.
In conventional histopathology, the most common brightfield stain applied to clinically important tissue sections is the hematoxylin and eosin (H&E) formulation. This method results in staining of nucleic acids and other so-called "basophilic" substances in the tissue section with a blue-purple coloration, and proteins and other "acidophilic" or "cosinophilic" tissue components with a pink coloration. This stain is used world-wide as a general screening method for the examination of all tissue components, to be followed in certain cases by special stains that have affinities for specific tissue elements such as microorganisms or nerve processes, and therefore enhance their appearance on stained tissue sections.
Methods have been introduced for en bloc staining, wherein the entire sample is stained by immersion before being subjected to infiltration and embedment. Sections are then cut from the block for transmission microscopy, or the cut face of the block itself is imaged in a process called block face microscopy or surface imaging microscopy. In the latter method, including that implemented in U.S. Pat. No. 4,960,330, a sample that has been stained en bloc with either conjugated or unconjugated fluorescent dyes is subsequently infiltrated by and embedded in a medium, commonly a plastic polymer, that is heavily opacified or otherwise treated to allow for the suppression of images of tissue originating from more than a small number of microns deep within the block. This results in the production of a thin, "virtual section" closely resembling a conventional glass-slide mounted tissue section.
Block face microscopy is advantageous over standard brightfield microscopy in that block face methods allow for the generation of high-quality microscopy images of biological tissue and other materials without the need to manufacture glass histology slides. The elimination of this requirement permits full automation of the histopathologic process, reducing incremental costs for each additional section produced, and consequently allowing for much greater amounts of information to be collected from each sample.
In block face microscopy, the digital virtual section as captured unmodified from the block face is a dark field image resulting from the colored emissions from the fluorescence-stained sample appearing against a black background representing the opacified polymer in which the sample is infiltrated and embedded. In contrast, conventional optical transmission microscopy, including that practiced in most surgical pathology laboratories and other medically-related microscopy-based diagnostic facilities, produces a brightfield image because thin slices of tissue and other material are stained with standard non-fluorescent dyes and are then trans-illuminated with a white or near-white light source, resulting in a background that is brighter, rather than darker than the tissue image.
In order to optimize block face microscopy images for clinical diagnosis and other purposes, it is preferable that the raw darkfield images captured from the face of the block be transformed and displayed as the more familiar images encountered in brightfield microscopy.